Integrated analysis of SR-like protein kinases Sky1 and Sky2 links signaling networks with transcriptional regulation in Candida albicans

Abstract

Fungal infections are a major global health burden where Candida albicans is among the most common fungal pathogen in humans and is a common cause of invasive candidiasis. Fungal phenotypes, such as those related to morphology, proliferation and virulence are mainly driven by gene expression, which is primarily regulated by kinase signaling cascades. Serine-arginine (SR) protein kinases are highly conserved among eukaryotes and are involved in major transcriptional processes in human and S. cerevisiae. Candida albicans harbors two SR protein kinases, while Sky2 is important for metabolic adaptation, Sky1 has similar functions as in S. cerevisiae. To investigate the role of these SR kinases for the regulation of transcriptional responses in C. albicans, we performed RNA sequencing of sky1Δ and sky2Δ and integrated a comprehensive phosphoproteome dataset of these mutants. Using a Systems Biology approach, we study transcriptional regulation in the context of kinase signaling networks. Transcriptomic enrichment analysis indicates that pathways involved in the regulation of gene expression are downregulated and mitochondrial processes are upregulated in sky1Δ. In sky2Δ, primarily metabolic processes are affected, especially for arginine, and we observed that arginine-induced hyphae formation is impaired in sky2Δ. In addition, our analysis identifies several transcription factors as potential drivers of the transcriptional response. Among these, a core set is shared between both kinase knockouts, but it appears to regulate different subsets of target genes. To elucidate these diverse regulatory patterns, we created network modules by integrating the data of site-specific protein phosphorylation and gene expression with kinase-substrate predictions and protein-protein interactions. These integrated signaling modules reveal shared parts but also highlight specific patterns characteristic for each kinase. Interestingly, the modules contain many proteins involved in fungal morphogenesis and stress response. Accordingly, experimental phenotyping shows a higher resistance to Hygromycin B for sky1Δ. Thus, our study demonstrates that a combination of computational approaches with integration of experimental data can offer a new systems biological perspective on the complex network of signaling and transcription. With that, the investigation of the interface between signaling and transcriptional regulation in C. albicans provides a deeper insight into how cellular mechanisms can shape the phenotype.

Keywords: Candida albicans; kinase signaling; network analysis; phosphoproteome; sky kinases; transcriptional regulation; transcriptome.

Figure 1

Transcriptional profiles of sky1Δ and sky2Δ compared to wild type reveals distinct responses for each kinase mutant. Volcano plots comparing transcriptomic abundance log₂FC changes (X-axis) and the adjusted P-values (Y-axis) for (A) sky1Δ and (B) sky2Δ versus wild type. RNA-Sequencing was performed after 4 hours growth of wild type, sky1Δ and sky2Δ in YPD medium. (C) Bar plot showing total number of differentially expressed (upper panel) and moderately to highly regulated genes (lower panel). Grouped bars represent upregulated (red), downregulated (blue) and total number of genes (grey) for sky1Δ and sky2Δ. (D) Venn diagrams of the total, downregulated and upregulated at least moderate differentially expressed genes (log₂FC ≥0.5 and adjusted P-value < 0.05) of sky1Δ and sky2Δ.

Figure 2

GO enrichment analysis reveals nuclear, mRNA processing and mitochondrial pathways for sky1Δ and glutamine and arginine metabolism pathways for sky2Δ. Bar plots show enriched biological processes (upper part; blue) and cellular components (lower part, brown) for (A) upregulated (moderate to high) as well as in (B) downregulated (moderate to high) genes. The X-axis represents the -log10 (adjusted P-value of enrichment) with the left side representing sky1Δ and the right side representing sky2Δ. For each GO category the total number of genes annotated in C albicans with this term is given in parentheses.

Figure 3

Arginine-induced wrinkle colony forming is impaired in the sky2Δ mutant. C. albicans strains were tested for arginine-induced filamentation on solid medium (0.17% YNB, 0.5% ammonium sulfate, 1% arginine [pH 6] and 0.17% YNB, 10 mM arginine, 0.2% glucose [pH 6]). The experiment was performed in duplicates and representative images were taken after 3 days of incubation at 37°C. The SKY2 complemented strain partially rescued the sky2Δ mutant phenotype. This was also shown in a previous study [(Brandt et al., 2022) for detailed data see Supplementary Figure 3 there].

Figure 4

Regulation and protein-protein interaction modules of enriched transcription factors point towards a few core regulators that are differentially phosphorylated or expressed in sky1Δ and sky2Δ. (A) Modules for sky1Δ and (B) sky2Δ enriched transcription factors (rectangles and phosphorylation sites (small circles) that are connected by simple edges representing a known direct regulation of the node, whereas double edges represent a known physical protein-protein interaction (PPI). Circle fill color intensity shows the strength of differential regulation or phosphorylation, where blue color show decreased, red increased and white no significant regulation (wild type vs skyΔ; P-value <0.05). Transcription factors are annotated with the relative frequency (percent) of regulated targets of the total significantly regulated genes.

Figure 5

Core set of transcription factors shared by sky1Δ and sky2Δ show distinct subsets of regulated genes in sky1Δ and sky2Δ and exhibit different phosphorylation patterns and interactors. (A) Venn diagrams for shared transcription factors represents the number of moderately expressed genes that were identified as regulation targets by transcription factor enrichment analysis. (B) Transcription factor network of sky1Δ (left) and sky2Δ (right) with transcription factors (rectangles), potential effector proteins (circles) and phosphorylation sites (small circles) connected by double edges representing a known physical protein-protein interaction. Circle fill color intensity shows the strength of differential regulation or phosphorylation, where blue color shows decreased, red increased and white no significant regulation (wild type vs skyΔ, P-value <0.05).

Figure 6

Integrated signaling modules of sky1Δ and sky2Δ. Network modules of (A) sky1Δ and (B) sky2Δ with kinases (octagons) and transcription factors (rectangles), phosphorylation sites (small circles) and first shell of potential effector proteins (large circles). Border color shows strongest phosphorylation change with significantly decreased (blue) and significantly increased phosphorylation (red; wild type vs skyΔ, P-value <0.05). Big node fill color intensity shows the strength of differential transcriptional regulation, where blue color shows down regulation, red upregulation and white no significant regulation (wild type vs skyΔ, P-value <0.05). Small node (phosphorylation site) fill color represents the strength of differential phosphorylation, with blue color showing decreased, red increased and white no significant regulation (wild type vs skyΔ, P-value <0.05). Kinase-substrate interactions (KSI) are represented by small doted (motif based only) and long doted (conserved only) edges with arrow. KSI supported by domain-domain interactions are shown by a continuous edge while experimentally described interaction are represented by double edges.

Figure 7

Deletion of SKY1 confers resistance to Hygromycin B and both Sky knockouts strains show distinct changes of the RAM-Pathway in transcriptomics and phosphoproteomics. (A) YPD overnight cultures of the strains were adjusted to an optical density (OD₆₀₀) of 1.0 and 5 µl of serial 10-fold dilutions and were spotted on YPD agar plates without or with 250 µg/ml or 300 µg/ml Hygromycin B and incubated for 3 days at 37°C. The experiment was performed in duplicates and representative images are shown. (B) Overview of the components of the RAM pathway of sky1Δ (left) and sky2Δ (right) with kinases (octagons) and transcription factors (rectangles), phosphorylation sites (small circles) and first shell of potential effector proteins (large circles). Big node fill color intensity shows the strength of differential transcriptional regulation, where blue color shows down regulation, red upregulation and grey no significant regulation (wild type vs skyΔ, P-value <0.05). Small node fill color represents the strength of differential phosphorylation, with blue color showing decreased, red increased and grey no significant regulation (wild type vs skyΔ, P-value <0.05).